This exploratory project is directed towards understanding the biological role of a novel oncogenic driver in Ewing sarcoma. Ewing sarcoma is an aggressive cancer of bone and soft tissues in children with poor long- term outcome. It is characterized by the chromosomal translocation generating a fusion oncogene between EWS and an Ets family transcription factor, most commonly FLI-1. EWS-FLI-1 translocation accounts for 85% of Ewing sarcoma cases. Since the cloning of the EWS-FLI-1 fusion oncogene, the predominant view in the Ewing sarcoma field has been that EWS-FLI-1 plays a central role in Ewing sarcomagenesis. EWS-FLI-1 is able to transform mouse cells such as NIH3T3 and C3H10T1/2 and the knockdown of EWS-FLI-1 inhibits the survival, proliferation and tumorigenicity of Ewing sarcoma cells, suggesting that EWS-FLI-1 is the causative oncogene. However, a variety of evidence also suggest that EWS-FLI-1 alone cannot fully explain the Ewing sarcomagenesis: 1) EWS-FLI-1 alone cannot transform any human cell types including human mesenchymal stem cells which are the putative cells of origin of Ewing sarcoma; 2) Generating a transgenic mouse model of Ewing sarcoma by using EWS-FLI-1 alone has been unsuccessful; and 3) Other genetic alterations such as mutations of INK4a and p53 confer worse clinical outcome. The applicant's group has identified a novel oncogenic driver for Ewing sarcoma, which is required for Ewing sarcoma proliferation and which cooperates with EWS-FLI-1 in mesenchymal stem cells. This project will address the biological role of this novel oncogenic driver in Ewing sarcoma by pursuing the following two specific aims: 1) Delineate its role in established Ewing sarcoma and 2) Modelling Ewing sarcoma by co- expression with EWS-FLI-1. The proposed research has the potential to shed new light on the long-standing conundrums in the Ewing sarcoma field such as the inability of EWS-FLI-1 to transform any human cell types, the failure to develop a genetic mouse model of Ewing sarcoma using EWS-FLI-1 alone, and the lack of a targeted therapy for Ewing sarcoma.